537 Trypanosomatid biodiversity in Costa Rica: genotyping of parasites from Heteroptera using the spliced leader RNA gene S. J. WESTENBERGER1,N.R.STURM1,D.YANEGA2,3,S.A.PODLIPAEV3#, R. ZELEDO´ N4,D.A.CAMPBELL1 and D. A. MASLOV5* 1 Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California – Los Angeles, Los Angeles, CA 90095-1487, USA 2 Department of Entomology and 3Entomology Research Museum, University of California – Riverside, Riverside, CA 92521, USA 3 Zoological Institute, Russian Academy of Sciences, 199034 St. Petersburg, Russia 4 School of Veterinary Medicine, National University, Heredia, Costa Rica 5 Department of Biology, University of California – Riverside, Riverside, CA 92521, USA (Received 31 January 2004; revised 6 April 2004; accepted 8 April 2004) SUMMARY The biodiversity of insect trypanosomes is largely unknown, resulting in significant gaps in the understanding of pathogen evolution. A culture-independent preliminary survey of trypanosomatid fauna was conducted for the parasites of Het- eroptera (Hemiptera) from several localities in Costa Rica. Trypanosomatid infections were detected by light microscopy of smeared gut contents. Out of 257 insects representing 6 families, infections were found in 62 cases; cultures were obtained for 29 new isolates. Gut material from infected hosts was preserved in the field using an SDS–EDTA buffer solution for subsequent DNA extraction in the laboratory. PCR amplification of the trypanosomatid-specific spliced leader (SL) RNA gene repeats was successful for 60 field samples. Eighteen distinct SL RNA typing units were identified in a set of 28 samples analysed in detail. Cluster analysis indicated that these typing units were unique and thus could represent new species and, in some cases, new genera. This study reveals only a minor fraction of the trypanosomatid biodiversity, which is anticipated to be high. Key words: Trypanosomatidae, biodiversity, mini-exon, SL RNA, genotyping. INTRODUCTION comprehensive biochemical and molecular analyses (Sbravate et al. 1989; Jankevicius et al. 1993; Trypanosomatid protozoa include several mono- Batistoti et al. 2001; Catarino et al. 2001). However, xenous (1 host) genera, including Blastocrithidia, cultivation is not possible for all of the known Crithidia, Herpetomonas, Leptomonas, Rhynchoido- trypanosomatids. Consequently, although there are monas and Wallaceina, that are often referred to as more than 400 published reports describing insect ‘insect trypanosomatids’ on the basis of their host. In trypanosomatids (Wallace, 1966; Podlipaev, 1990), contrast to the insect-transmitted dixenous (2 hosts) the identity of most of these organisms remains parasites of vertebrates from the genera Trypanosoma uncertain. and Leishmania, very little is known about the di- Insect trypanosomatids are broadly distributed. versity, phylogeny, host specificity and geographical Two insect orders, Hemiptera and Diptera, account distribution of insect trypanosomatids. Thus our for nearly 35 and 55%, respectively, of the known knowledge of the entire family remains fragmentary. hosts (Podlipaev, 1990). A recent 18S rRNA phylo- The lack of sufficient attention to the monoxenous genetic study (Merzlyak et al. 2001) identified several parasites is only partially responsible for this situ- previously undescribed lineages of insect trypano- ation. Insect trypanosomatids are usually difficult to somatids (Hollar, Lukesˇ & Maslov, 1998), and led to distinguish from each other using light microscopy. the suggestion that the group is extremely diverse An ideal solution to this problem would be the re- (Stevens, 2001). covery of each new isolate in axenic culture, serving Application of PCR is a viable alternative to both as a species voucher and a source of material for cultivation in microbial biodiversity surveys, as a minimal amount of genetic material is sufficient * Corresponding author: Department of Biology, Uni- for genotyping, such as might be contained in a versity of California – Riverside, 3401 Watkins Drive, field sample. The main advantages of a culture- Riverside, CA 92521, USA. Tel: +1 909 787 6485. Fax: +1 909 787 4286. E-mail: [email protected] independent approach are that it allows for identifi- # Deceased. cation of organisms that are difficult or impossible Parasitology (2004), 129, 537–547. f 2004 Cambridge University Press DOI: 10.1017/S003118200400592X Printed in the United Kingdom S. J. Westenberger and others 538 to cultivate and avoids culture-induced homogen- dissected using disposable fine-point wooden tooth- ization of mixed infections. Culture-independent picks. After opening the abdomen, the contents were surveys are rapidly expanding the investigated range suspended in a few drops (0.2–0.3 ml) of 1rSSC of eukaryotic biodiversity (Baldauf, 2003) including buffer (150 mM NaCl, 15 mM sodium citrate) on a the discovery of potentially deep branching free- microscope slide. The slides were examined using living kinetoplastids (Lopez-Garcia et al. 2003). phase-contrast light microscopy at 400r magnifi- The multicopy spliced leader (SL) RNA (also re- cation with a cover-slip. Upon detection of a trypa- ferred to as mini-exon donor RNA) gene array nosomatid infection, the material from the slide was represents one of the best studied and most useful divided between two 1.5 ml Eppendorf tubes: one markers for genotyping of trypanosomatids (Murthy, tube containing 1 ml of 1% SDS, 0.1 M EDTA for Dibbern & Campbell, 1992); its highly conserved DNA preservation, and another tube with 1 ml of exon sequence allows for specific and efficient am- brain heart infusion (BHI) medium, supplemented plification from a broad selection of species, a mod- with 10 mg/ml haemin, 100 mg/ml ampicillin, 100 mg/ erately variable intron is useful for selective grouping ml tetracycline and 50 mg/ml chloramphenicol to of related species, while a hypervariable intergenic initiate a primary culture. The remaining material region provides the highest level of discrimination on the slide was fixed with ethanol and air-dried among isolates. In addition, SL RNA is not present for subsequent staining with Giemsa. The post- in the insect or vertebrate hosts of trypanosomatids. dissection insects (‘xenotypes’) were deposited in SL RNA genotyping has been used to investigate the UCR Research Entomology Museum as voucher relationships within the genera Crithidia (Fernandes specimens. et al. 1997), Herpetomonas, Leptomonas and Blasto- crithidia (Podlipaev et al. 2004), Phytomonas (Sturm, Sample handling: laboratory stage Fernandes & Campbell, 1995; Dollet, Sturm & Campbell, 2001), Leishmania and Endotrypanum Extraction of DNA from the material preserved (Fernandes, Degrave & Campbell, 1993; Fernandes in the SDS–EDTA buffer was performed after et al. 1994), and Trypanosoma (Souto et al. 1996; digestion with pronase (200 mg/ml) at 65 xC for Fernandes et al. 1998; Grisard, Campbell & 30 min. The lysate was extracted with Tris-buffered Romanha, 1999; Grisard, Sturm & Campbell, 2003). phenol (pH 8), followed by extraction with phenol– Development of group-specific amplification proto- chloroform. Nucleic acid from the aqueous phase cols and hybridization probes has further increased was precipitated with isopropanol after addition of the versatility of the SL RNA gene marker (Ramos glycogen (5 mg/ml) as a carrier. The pellets were et al. 1996; Teixeira et al. 1996; Fernandes et al. washed with ethanol and resuspended in 100 mlof 1997; Harris et al. 1998; Serrano et al. 1999; 10 mM Tris–HCl, pH 8.0, 0.1mM EDTA. Fernandes et al. 2001; Godoi et al. 2002; Marfurt et al. 2003), such that it is a logical choice for con- PCR amplification, cloning and sequencing ducting culture-independent surveys of trypanoso- matids. The SL RNA gene repeats were amplified from Our long-term goal is to describe the full range of sample DNAs using the overlapping oligonucleo- diversity among insect trypanosomatids. In this work tides M167, 5k-gggaagcTTCTGTACT(A/T)TAT we apply a SL RNA gene repeat-based PCR strategy TGGTA, and M168, 5k-gggaattCAATA(A/T)AGT for a survey of trypanosomatids from Heteroptera ACAGAAACTG, wherein the lower case letters (Hemiptera) in Costa Rica, the country known for its indicate nucleotides added to the genomic sequence great insect diversity and a relative proximity of to facilitate PCR. These oligonucleotides were based multiple ecosystems. on the exon-specific primers suggested originally (Murthy et al. 1992). The DNA contained in 1 mlof each sample was used for PCR with Taq polymerase MATERIALS AND METHODS with the following cycling profile: initial denatura- tion at 95 xC for 5 min, followed by 3 low-stringency Sample handling: field stage cycles (95 xC for 30 s, 42 xC for 1 min, 65 xC for Heteropteran bugs were collected at 5 sites in Costa 2 min 30 s) and 32 high-stringency cycles (95 xC for Rica: 2 locations in the Carara National Park, near 30 s, 50 xC for 1 min, 72 xC for 2 min 30 s). Agarose the northern boundary of the Guanacaste National gel electrophoresis was performed by standard pro- Park, near the La Selva Biological Station, and at the cedures (Sambrook, Fritsch & Maniatis, 1989). The western boundary of the Braulio Carrillo National Phytomonas-specific hybridization oligonucleotide Park (El Ceibo). Insects were collected by sweep- probe (‘probe 42’) was 5k-TTGGACTCGGGGC- netting, attraction to black and mercury vapour light, CTTCGG. The DNA blots were hybridized in or by hand picking. Live specimens were kept in- 6rSSC